Triazolo-pyridazine derivatives as ligands for gaba receptors

ABSTRACT

1,2,4-triazolo[4,3-b]pyridazine derivatives, possessing a difluoro-or tri-fluoro-substituted phenyl ring at the 3-position, a triazolyl moiety at the 6-position, and a tert-butyl group at the 7-position, are selective ligands for GABA A  receptors, in particular having high affinity for the α2 and/or α3 subunit thereof, and are useful in the treatment of anxiety and convulsions.

This application is a 371 of PCT/GB00/00183 and claims priority fromGreat Britain Application No. 9912429.9, filed May 27, 1999, GreatBritain Application No. 9901744.4, filed Jan. 27, 1999, and GreatBritain Application No. 9901743.6, filed Jan. 27, 1999.

BACKGROUND OF THE INVENTION

The present invention relates to a class of substitutedtriazolo-pyridazine derivatives and to their use in therapy. Moreparticularly, this invention is concerned with substituted1,2,4-triazolo[4,3-b]pyridazine derivatives which are ligands forGABA_(A) receptors and are therefore useful in the therapy ofdeleterious mental states.

Receptors for the major inhibitory neurotransmitter, gamma-aminobutyricacid (GABA), are divided into two main classes: (1) GABA_(A) receptors,which are members of the ligand-gated ion channel superfamily; and (2)GABA_(B) receptors, which may be members of the G-protein linkedreceptor superfamily. Since the first cDNAs encoding individual GABA_(A)receptor subunits were cloned the number of known members of themammalian family has grown to include at least six α subunits, four βsubunits, three γ subunits, one δ subunit, one ε subunit and two ρsubunits.

Although knowledge of the diversity of the GABA_(A) receptor gene familyrepresents a huge step forward in our understanding of this ligand-gatedion channel, insight into the extent of subtype diversity is still at anearly stage. It has been indicated that an α subunit, a β subunit and aγ subunit constitute the minimum requirement for forming a fullyfunctional GABA_(A) receptor expressed by transiently transfecting cDNAsinto cells. As indicated above, δ, ε and ρ subunits also exist, but arepresent only to a minor extent in GABA_(A) receptor populations.

Studies of receptor size and visualisation by electron microscopyconclude that, like other members of the ligand-gated ion channelfamily, the native GABA_(A) receptor exists in pentameric form. Theselection of at least one α, one β and one γ subunit from a repertoireof seventeen allows for the possible existence of more than 10,000pentameric subunit combinations. Moreover, this calculation overlooksthe additional permutations that would be possible if the arrangement ofsubunits around the ion channel had no constraints (i.e. there could be120 possible variants for a receptor composed of five differentsubunits).

Receptor subtype assemblies which do exist include, amongst many others,α1β2γ2, α2β2/3γ2, α3βγ2/3, α2βγ1, α5β3γ2/3, α6βγ2, α6βδ and α4βδ.Subtype assemblies containing an al subunit are present in most areas ofthe brain and are thought to account for over 40% of GABA_(A) receptorsin the rat. Subtype assemblies containing α2 and α3 subunitsrespectively are thought to account for about 25% and 17% of GABA_(A)receptors in the rat. Subtype assemblies containing an α5 subunit areexpressed predominantly in the hippocampus and cortex and are thought torepresent about 4% of GABA_(A) receptors in the rat.

A characteristic property of all known GABA_(A) receptors is thepresence of a number of modulatory sites, one of which is thebenzodiazepine (BZ) binding site. The BZ binding site is the mostexplored of the GABA_(A) receptor modulatory sites, and is the sitethrough which anxiolytic drugs such as diazepam and temazepam exerttheir effect. Before the cloning of the GABA_(A) receptor gene family,the benzodiazepine binding site was historically subdivided into twosubtypes, BZ1 and BZ2, on the basis of radioligand binding studies. TheBZ1 subtype has been shown to be pharmacologically equivalent to aGABA_(A) receptor comprising the α1 subunit in combination with a βsubunit and γ2. This is the most abundant GABA_(A) receptor subtype, andis believed to represent almost half of all GABA_(A) receptors in thebrain.

Two other major populations are the α2βγ2 and α3βγ2/3 subtypes. Togetherthese constitute approximately a further 35% of the total GABA_(A)receptor repertoire. Pharmacologically this combination appears to beequivalent to the BZ2 subtype as defined previously by radioligandbinding, although the BZ2 subtype may also include certain α5-containingsubtype assemblies. The physiological role of these subtypes hashitherto been unclear because no sufficiently selective agonists orantagonists were known.

It is now believed that agents acting as BZ agonists at α1βγ2, α2βγ2 orα3βγ2 subunits will possess desirable anxiolytic properties. Compoundswhich are modulators of the benzodiazepine binding site of the GABA_(A)receptor by acting as BZ agonists are referred to hereinafter as“GABA_(A) receptor agonists”. The α1-selective GABA_(A) receptoragonists alpidem and zolpidem are clinically prescribed as hypnoticagents, suggesting that at least some of the sedation associated withknown anxiolytic drugs which act at the BZ1 binding site is mediatedthrough GABA_(A) receptors containing the al subunit. Accordingly, it isconsidered that GABA_(A) receptor agonists which interact morefavourably with the α2 and/or α3 subunit than with al will be effectivein the treatment of anxiety with a reduced propensity to cause sedation.Also, agents which are antagonists or inverse agonists at α1 might beemployed to reverse sedation or hypnosis caused by al agonists.

The compounds of the present invention, being selective ligands forGABA_(A) receptors, are therefore of use in the treatment and/orprevention of a variety of disorders of the central nervous system. Suchdisorders include anxiety disorders, such as panic disorder with orwithout agoraphobia, agoraphobia without history of panic disorder,animal and other phobias including social phobias, obsessive-compulsivedisorder, stress disorders including post-traumatic and acute stressdisorder, and generalized or substance-induced anxiety disorder;neuroses; convulsions; migraine; depressive or bipolar disorders, forexample single-episode or recurrent major depressive disorder, dysthymicdisorder, bipolar I and bipolar II manic disorders, and cyclothymicdisorder; psychotic disorders including schizophrenia; neurodegenerationarising from cerebral ischemia; attention deficit hyperactivitydisorder; and disorders of circadian rhythm, e.g. in subjects sufferingfrom the effects of jet lag or shift work.

Further disorders for which selective ligands for GABA_(A) receptors maybe of benefit include pain and nociception; emesis, including acute,delayed and anticipatory emesis, in particular emesis induced bychemotherapy or radiation, as well as post-operative nausea andvomiting; eating disorders including anorexia nervosa and bulimianervosa; premenstrual syndrome; muscle spasm or spasticity, e.g. inparaplegic patients; and hearing loss. Selective ligands for GABA_(A)receptors may also be effective as pre-medication prior to anaesthesiaor minor procedures such as endoscopy, including gastric endoscopy.

WO 98/04559 describes a class of substituted and 7,8-ring fused1,2,4-triazolo[4,3-b]pyridazine derivatives which are stated to beselective ligands for GABA_(A) receptors beneficial in the treatmentand/or prevention of neurological disorders including anxiety andconvulsions.

SUMMARY OF THE INVENTION

The present invention provides a class of triazolo-pyridazinederivatives which possess desirable binding properties at variousGABA_(A) receptor subtypes. The compounds in accordance with the presentinvention have good affinity as ligands for the α2 and/or α3 subunit ofthe human GABA_(A) receptor. The compounds of this invention interactmore favourably with the α2 and/or α3 subunit than with the α1 subunit.Indeed, the compounds of the invention exhibit functional selectivity interms of a selective efficacy for the α2 and/or α3 subunit relative tothe α1 subunit.

The compounds of the present invention are GABA_(A) receptor subtypeligands having a binding affinity (K_(i)) for the α2 and/or α3 subunit,as measured in the assay described hereinbelow, of less than 1 nM.Furthermore, the compounds in accordance with this invention exhibitfunctional selectivity in terms of a selective efficacy for the α2and/or α3 subunit relative to the α1 subunit. Moreover, the compoundsaccording to the present invention possess interesting pharmacokineticproperties, notably in terms of improved oral bioavailability.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound of formula I, or apharmaceutically acceptable salt thereof:

wherein

Y represents hydrogen and Z represents fluoro, or Y represents fluoroand Z represents hydrogen or fluoro; and

R¹ represents methyl or ethyl.

The compounds in accordance with the present invention are encompassedwithin the generic scope of WO 98/04559. There is, however, no specificdisclosure therein of compounds corresponding to those of formula I asdefined above.

For use in medicine, the salts of the compounds of formula I above willbe pharmaceutically acceptable salts. Other salts may, however, beuseful in the preparation of the compounds of formula I or of theirpharmaceutically acceptable salts. Suitable pharmaceutically acceptablesalts of the compounds of formula I include acid addition salts whichmay, for example, be formed by mixing a solution of the compound offormula I with a solution of a pharmaceutically acceptable acid such ashydrochloric acid, sulphuric acid, methanesulphonic acid, fumaric acid,maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid,citric acid, tartaric acid, carbonic acid or phosphoric acid.

The present invention also provides a compound of formula I as depictedabove, or a pharmaceutically acceptable salt thereof, wherein Y and Zboth represent fluoro; and R¹ represents methyl or ethyl.

A particular sub-class of the compounds in accordance with the inventionis represented by the compounds of formula IA, and pharmaceuticallyacceptable salts thereof:

wherein Y, Z and R¹ are as defined above.

Specific sub-classes of the compounds in accordance with the inventionare represented by the compounds of formula IIA, IIB, and IIC, andpharmaceutically acceptable salts thereof.

wherein R¹ is as defined above.

In one embodiment of the compounds according to the invention, themoiety R¹ represents methyl.

In another embodiment of the compounds according to the invention, themoiety R¹ represents ethyl.

Specific compounds within the scope of the present invention include:

3-(2,5-difluorophenyl)-7-(1,1-dimethylethyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;

3-(2,5-difluorophenyl)-7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;

3-(2,6-difluorophenyl)-7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine;

7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2,3,6-trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine;

7-(1,1-dimethylethyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2,3,6-trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine;

7-(1,1-dimethylethyl)-6-(1-methyl-1H-1,2,4-triazol-3-ylmethoxy)-3-(2,3,6-trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine;

and pharmaceutically acceptable salts thereof.

Also provided by the present invention is a method for the treatmentand/or prevention of anxiety which comprises administering to a patientin need of such treatment an effective amount of a compound of formula Ias defined above or a pharmaceutically acceptable salt thereof.

Further provided by the present invention is a method for the treatmentand/or prevention of convulsions (e.g. in a patient suffering fromepilepsy or a related disorder) which comprises administering to apatient in need of such treatment an effective amount of a compound offormula I as defined above or a pharmaceutically acceptable saltthereof.

The binding affinity (K_(i)) of the compounds according to the presentinvention for the α3 subunit of the human GABA_(A) receptor isconveniently as measured in the assay described hereinbelow. The α3subunit binding affinity (K_(i)) of the compounds of the invention isless than 1 nM.

The compounds according to the present invention elicit a selectivepotentiation of the GABA EC₂₀ response in stably transfected recombinantcell lines expressing the α3 subunit of the human GABA_(A) receptorrelative to the potentiation of the GABA EC₂₀ response elicited instably transfected recombinant cell lines expressing the α1 subunit ofthe human GABA_(A) receptor.

The potentiation of the GABA EC₂₀ response in stably transfected celllines expressing the α3 and α1 subunits of the human GABA_(A) receptorcan conveniently be measured by procedures analogous to the protocoldescribed in Wafford et al., Mol. Pharmacol., 1996, 50, 670-678. Theprocedure will suitably be carried out utilising cultures of stablytransfected eukaryotic cells, typically of stably transfected mouse Ltk⁻fibroblast cells.

The compounds according to the present invention exhibit anxiolyticactivity, as may be demonstrated by a positive response in the elevatedplus maze and conditioned suppression of drinking tests (cf. Dawson etal., Psychopharmacology, 1995, 121,109-117). Moreover, the compounds ofthe invention are substantially non-sedating, as may be confirmed by anappropriate result obtained from the response sensitivity(chain-pulling) test (cf. Bayley et al., J. Psychopharmacol., 1996, 10,206-213).

The compounds according to the present invention may also exhibitanticonvulsant activity. This can be demonstrated by the ability toblock pentylenetetrazole-induced seizures in rats and mice, following aprotocol analogous to that described by Bristow et al. in J. Pharmacol.Exp. Ther., 1996, 279, 492-501.

Since they elicit behavioural effects, the compounds of the inventionplainly are brain-penetrant; in other words, these compounds are capableof crossing the so-called “blood-brain barrier”. Advantageously, thecompounds of the invention are capable of exerting their beneficialtherapeutic action following administration by the oral route.

The invention also provides pharmaceutical compositions comprising oneor more compounds of this invention in association with apharmaceutically acceptable carrier. Preferably these compositions arein unit dosage forms such as tablets, pills, capsules, powders,granules, sterile parenteral solutions or suspensions, metered aerosolor liquid sprays, drops, ampoules, auto-injector devices orsuppositories; for oral, parenteral, intranasal, sublingual or rectaladministration, or for administration by inhalation or insufflation. Forpreparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical carrier, e.g. conventionaltableting ingredients such as corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother pharmaceutical diluents, e.g. water, to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention, or a pharmaceutically acceptable saltthereof. When referring to these preformulation compositions ashomogeneous, it is meant that the active ingredient is dispersed evenlythroughout the composition so that the composition may be readilysubdivided into equally effective unit dosage forms such as tablets,pills and capsules. This solid preformulation composition is thensubdivided into unit dosage forms of the type described above containingfrom 0.1 to about 500 mg of the active ingredient of the presentinvention. Typical unit dosage forms contain from 1 to 100 mg, forexample 1, 2, 5, 10, 25, 50 or 100 mg, of the active ingredient. Thetablets or pills of the novel composition can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permits theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavoured syrups, aqueous or oilsuspensions, and flavoured emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles. Suitable dispersing or suspendingagents for aqueous suspensions include synthetic and natural gums suchas tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose,methylcellulose, polyvinyl-pyrrolidone or gelatin.

In the treatment of anxiety, a suitable dosage level is about 0.01 to250 mg/kg per day, preferably about 0.05 to 100 mg/kg per day, andespecially about 0.05 to 5 mg/kg per day. The compounds may beadministered on a regimen of 1 to 4 times per day.

The compounds of formula I as defined above may be prepared by a processwhich comprises reacting a compound of formula III with a compound offormula IV:

wherein Y, Z and R¹ are as defined above, and L¹ represents a suitableleaving group.

The leaving group L¹ is typically a halogen atom, especially chloro.

The reaction between compounds III and IV is conveniently effected bystirring the reactants in a suitable solvent, in the presence of a base.Typically, the solvent is N,N-dimethylformamide, and the base is astrong base such as sodium hydride. In one preferred embodiment, thesolvent is dimethylsulfoxide, and the base is caesium carbonate. Inanother preferred embodiment, the solvent is 1-methyl-2-pyrrolidinone,and the base is sodium hydroxide, in which case the reaction isadvantageously performed at a temperature in the region of 0° C.

The intermediates of formula III above may be prepared by reacting acompound of formula V with a substantially equimolar amount of ahydrazine derivative of formula VI:

wherein Y, Z and L¹ are as defined above, and L² represents a suitableleaving group; followed, if necessary, by separation of the resultingmixture of isomers by conventional means.

The leaving group L² is typically a halogen atom, especially chloro. Inthe intermediates of formula V, the leaving groups L¹ and L² may be thesame or different, but are suitably the same, preferably both chloro.

The reaction between compounds V and VI is conveniently effected byheating the reactants in the presence of a proton source such astriethylamine hydrochloride, typically at reflux in an inert solventsuch as xylene or 1,4-dioxane.

Alternatively, the intermediates of formula III above may be prepared byreacting a hydrazine derivative of formula VII with an aldehydederivative of formula VIII:

wherein Y, Z and L¹ are as defined above; followed by cyclization of theintermediate Schiff's base thereby obtained.

The reaction between compounds VII and VIII is conveniently effectedunder acidic conditions, for example in the presence of a mineral acidsuch as hydrochloric acid. Cyclization of the resulting Schiff's baseintermediate may then conveniently be carried out by treatment withiron(III) chloride in a suitable solvent, e.g. an alcoholic solvent suchas ethanol, at an elevated temperature, typically at a temperature inthe region of 80° C.

The intermediates of formula VII above may be prepared by reacting theappropriate compound of formula V as defined above with hydrazinehydrate, typically in isobutyl alcohol at an elevated temperature, e.g.a temperature in the region of 90° C., or in 1,4-dioxane or ethanol atthe reflux temperature of the solvent; followed, if necessary, byseparation of the resulting mixture of isomers by conventional means.

In an alternative approach, the intermediates of formula III above maybe prepared by reacting the hydrazine derivative of formula VII asdefined above with a compound of formula IX:

wherein Y and Z are as defined above, and Q represents a reactivecarboxylate moiety; followed, if necessary, by cyclization of thehydrazide derivative of formula X thereby obtained:

wherein Y, Z and L¹ are as defined above.

Suitable values for the reactive carboxylate moiety Q include esters,for example C₁₋₄ alkyl esters; acid anhydrides, for example mixedanhydrides with C₁₋₄ alkanoic acids; acid halides, for example acidchlorides; and acylimidazoles. Suitably, Q represents an acid chloridemoiety.

The reaction between compounds VII and IX is conveniently effected byheating in a solvent such as 1-methyl-2-pyrrolidinone to a temperaturetypically in the region of 160° C.

Alternatively, the reaction between compounds VII and IX may be effectedunder basic conditions, e.g. in the presence of triethylamine, suitablyin an inert solvent such as diethyl ether, and typically at atemperature in the region of 0° C. Cyclization of the resulting compoundof formula X may then conveniently be carried out by treatment with1,2-dibromo-1,1,2,2-tetrachloroethane and triphenylphosphine, in thepresence of a base such as triethylamine, suitably in an inert solventsuch as acetonitrile, and typically at a temperature in the region of 0°C.

The reaction between compound V and hydrazine hydrate or compound VIwill, as indicated above, possibly give rise to a mixture of isomericproducts depending upon whether the hydrazine nitrogen atom displacesthe leaving group L¹ or L². Thus, in addition to the required product offormula III, the isomeric compound wherein the hydrazine moietydisplaces the leaving group L¹ will possibly be obtained to some extent;and likewise for compound VII. For this reason it might be necessary toseparate the resulting mixture of isomers by conventional methods suchas chromatography.

In another procedure, the compounds of formula I as defined above may beprepared by a process which comprises reacting a compound of formula XI(or its 1,2,4-triazolo[4,3-b]pyridazin-6-one tautomer) with a compoundof formula XII:

wherein Y, Z and R¹ are as defined above, and L³ represents a suitableleaving group.

The leaving group L³ is suitably a halogen atom, typically chloro orbromo.

The reaction between compounds XI and XII is conveniently effected bystirring the reactants in a suitable solvent, typicallyN,N-dimethylformamide, in the presence of a strong base such as sodiumhydride.

The intermediate of formula XI above may conveniently be prepared byreacting a compound of formula III as defined above with an alkali metalhydroxide, e.g. sodium hydroxide. The reaction is conveniently effectedin an inert solvent such as aqueous 1,4-dioxane, ideally at the refluxtemperature of the solvent.

In a further procedure, the compounds of formula I as defined above maybe prepared by a process which comprises reacting trimethylacetic acidwith a compound of formula XIII:

wherein Y, Z and R¹ are as defined above; in the presence of silvernitrate and ammonium persulphate.

The reaction is conveniently carried out in a suitable solvent, forexample water or aqueous acetonitrile, optionally under acidicconditions, e.g. using trifluoroacetic acid or sulphuric acid, typicallyat an elevated temperature.

The intermediates of formula XIII correspond to the compounds of formulaI as defined above wherein the tert-butyl substituent at the 7-positionis absent, and they may therefore be prepared by methods analogous tothose described above for preparing the corresponding compounds offormula I.

In a still further procedure, the compounds of formula I as definedabove may be prepared by a process which comprises reacting a compoundof formula XIV with a compound of formula XV:

wherein Y, Z and R¹ are as defined above, M represents —B(OH)₂ or—Sn(Alk)₃ in which Alk represents a C₁₋₆ alkyl group, typically n-butyl,and L⁴ represents a suitable leaving group; in the presence of atransition metal catalyst.

The leaving group L⁴ is suitably a halogen atom, e.g. bromo.

A suitable transition metal catalyst of use in the reaction betweencompounds XIV and XV comprisesdichlorobis(triphenylphosphine)palladium(II) ortetrakis(triphenylphosphine)palladium(0).

The reaction between compounds XIV and XV is conveniently effected in aninert solvent such as N,N-dimethylformamide, typically at an elevatedtemperature.

The intermediates of formula XIV may be prepared by reacting a compoundof formula IV as defined above with a compound of formula XVI:

wherein L¹ and L⁴ are as defined above; under conditions analogous tothose described above for the reaction between compounds III and IV.

Where R¹ is methyl, the relevant intermediate of formula IV above may beprepared by the procedures described in EP-A-0421210, or by methodsanalogous thereto. Where R¹ is ethyl, the relevant intermediate offormula IV may conveniently be prepared by the method described in theaccompanying Examples.

The intermediates of formula V above may be prepared by reactingtrimethylacetic acid with a compound of formula XVII:

wherein L¹ and L² are as defined above; in the presence of silvernitrate and ammonium persulphate; under conditions analogous to thosedescribed above for the reaction between trimethylacetic acid andcompound XIII.

Where they are not commercially available, the starting materials offormula VI, VIII, IX, XII, XV, XVI and XVII may be prepared by methodsanalogous to those described in the accompanying Examples, or bystandard methods well known from the art.

During any of the above synthetic sequences it may be necessary and/ordesirable to protect sensitive or reactive groups on any of themolecules concerned. This may be achieved by means of conventionalprotecting groups, such as those described in Protective Groups inOrganic Chemistry, ed. J. F. W. McOmie, Plenum Press, 1973; and T. W.Greene & P. G. M. Wuts, Protective Groups in Organic Synthesis, JohnWiley & Sons, 1991. The protecting groups may be removed at a convenientsubsequent stage using methods known from the art.

The following Examples illustrate the preparation of compounds accordingto the invention.

The compounds in accordance with this invention potently inhibit thebinding of [³H]-flumazenil to the benzodiazepine binding site of humanGABA_(A) receptors containing the α2 or α3 subunit stably expressed inLtk⁻ cells.

Reagents

Phosphate buffered saline (PBS).

Assay buffer: 10 mM KH₂PO₄, 100 mM KCl, pH 7.4 at room temperature.

[³H]-Flumazenil (18 nM for α1β3γ2 cells; 18 nM for α2β3γ2 cells; 10 nMfor α3β3γ2 cells) in assay buffer.

Flunitrazepam 100 μM in assay buffer.

Cells resuspended in assay buffer (1 tray to 10 ml).

Harvesting Cells

Supernatant is removed from cells. PBS (approximately 20 ml) is added.The cells are scraped and placed in a 50 ml centrifuge tube. Theprocedure is repeated with a further 10 ml of PBS to ensure that most ofthe cells are removed. The cells are pelleted by centrifuging for 20 minat 3000 rpm in a benchtop centrifuge, and then frozen if desired. Thepellets are resuspended in 10 ml of buffer per tray (25 cm×25 cm) ofcells.

Assay

Can be carried out in deep 96-well plates or in tubes. Each tubecontains:

300 μl of assay buffer.

50 μl of [³H]-flumazenil (final concentration for α1β3γ2: 1.8 nM; forα2β3γ2: 1.8 nM; for α3β3γ2: 1.0 nM).

50 μl of buffer or solvent carrier (e.g. 10% DMSO) if compounds aredissolved in 10% DMSO (total); test compound or flunitrazepam (todetermine non-specific binding), 10 μM final concentration.

100 μl of cells.

Assays are incubated for 1 hour at 40° C., then filtered using either aTomtec or Brandel cell harvester onto GF/B filters followed by 3×3 mlwashes with ice cold assay buffer. Filters are dried and counted byliquid scintillation counting. Expected values for total binding are3000-4000 dpm for total counts and less than 200 dpm for non-specificbinding if using liquid scintillation counting, or 1500-2000 dpm fortotal counts and less than 200 dpm for non-specific binding if countingwith meltilex solid scintillant. Binding parameters are determined bynon-linear least squares regression analysis, from which the inhibitionconstant K_(i) can be calculated for each test compound.

The compounds of the accompanying Examples were tested in the aboveassay, and all were found to possess a K_(i) value for displacement of[³H]-flumazenil from the α2 and/or α3 subunit of the human GABA_(A)receptor of less than 1 nM.

EXAMPLE 13-(2,5-Difluorophenyl)-7-(1,1-dimethylethyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine

a) 3,6-Dichloro-4-(1,1-dimethylethyl)pyridazine

Concentrated sulfuric acid (53.6 ml, 1.0 mol) was added carefully to astirred suspension of 3,6-dichloropyridazine (50.0 g, 0.34 mol) in water(1.25 l). This mixture was then heated to 70° C. (internal temperature)before the addition of trimethylacetic acid (47.5 ml, 0.41 mol). Asolution of silver nitrate (11.4 g, 0.07 mol) in water (20 ml) was thenadded over approximately one minute. This caused the reaction mixture tobecome milky in appearance. A solution of ammonium persulphate (230 g,1.0 mol) in water (0.63 l) was then added over 20-30 minutes. Theinternal temperature rose to approximately 85° C. During the additionthe product formed as a sticky precipitate. Upon complete addition thereaction was stirred for an additional 10 minutes, then allowed to coolto room temperature. The mixture was then poured onto ice and basifiedwith concentrated aqueous ammonia, with the addition of more ice asrequired to keep the temperature below 10° C. The aqueous was extractedwith dichloromethane (3×300 ml). The combined extracts were dried(MgSO₄), filtered and evaporated to give 55.8 g of crude product as anoil. This was purified by silica gel chromatography using 0-15% ethylacetate in hexane as eluent to give 37.31 g (53%) of the desiredcompound. Data for the title compound: ¹H NMR (360 MHz, d₆-DMSO) δ1.50(9H, s), 7.48 (1H, s); MS (ES⁺) m/e 205 [MH]⁺, 207 [MH]⁺.

b) 3-Chloro-4-(1,1-dimethylethyl)-6-hydrazinylpyridazine

To a solution of 3,6-dichloro-4-(1,1-dimethylethyl)pyridazine (2.0 g,9.76 mmol) in ethanol (30 ml) was added hydrazine hydrate (0.34 ml, 10.9mmol) dropwise. The reaction mixture was heated at reflux for 18 h underan atmosphere of nitrogen. The solvent was removed under high vacuum toleave a residue to which was added 5N hydrochloric acid (50 ml). Thesolution obtained was washed with dichloromethane (20 ml) and theaqueous layer was poured on to a mixture of ice and aqueous ammonia. Theresultant solid was collected by filtration and dried under vacuum toyield the title compound (1.2 g). Data for the title compound: ¹H NMR(360 MHz, DMSO) δ1.39 (3H, t, J=7.3 Hz), 4.35 (2H), 7.07 (1H, s), 8.07(1H, s); MS (ES⁺) m/e 201, 203 [MH]⁺.

c)6-Chloro-3-(2,5-difluorophenyl)-7-(1,1-dimethylethyl)-1,2,4-triazolo[4,3-b]pyridazine

To a slurry of 3-chloro-4-(1,1-dimethylethyl)-6-hydrazinylpyridazine(1.3 g, 6.5 mmol) in 0.1N hydrochloric acid (60 ml) was added2,5-difluorobenzaldehyde (0.70 ml, 6.5 mmol) and the reaction mixturewas stirred at room temperature for 30 minutes and then heated to 60° C.for 40 minutes. The reaction mixture was allowed to cool and theresultant solid was collected by filtration, dried and dissolved inethanol (60 ml). To this solution was added iron(III) chloridehexahydrate (5.4 g, 32.5 mmol) in ethanol (15 ml) dropwise over 10minutes at 80° C. The reaction mixture was stirred at 80° C. for 2 h,allowed to cool and the solvent removed by evaporation under vacuum. Theresidue was dissolved in dichloromethane (100 ml) and washed with water(3×100 ml), brine, dried (MgSO₄), filtered and concentrated under vacuumto yield the title compound (0.75 g). Data for the title compound: ¹HNMR (250 MHz, CDCl₃) δ1.57 (9H, s), 7.22-7.29 (2H, m), 7.65-7.71 (1H,m), 8.19 (1H, s); MS (ES⁺) m/e 323 [MH]⁺.

d)3-(2,5-Difluorophenyl)-7-(1,1-dimethylethyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine

To a solution of (2-methyl-2H-1,2,4-triazol-3-yl)methanol (0.069 g, 0.4mmol) and6-chloro-3-(2,5-difluorophenyl)-7-(1,1-dimethylethyl)-1,2,4-triazolo[4,3-b]pyridazine(0.10 g, 0.31 mmol) in DMF (10 ml) was added sodium hydride (0.015 g ofa 60% dispersion in oil, 1.2 mol eq.) and the reaction mixture wasstirred at room temperature for 40 minutes. After this time the reactionmixture was diluted with water (80 ml) and the solid that precipitatedwas collected by filtration and washed several times with water in thesinter funnel. The solid was recrystallised from ethyl acetate/hexane togive pure title compound (0.088 g, 65%). Data for the title compound: ¹HNMR (360 MHz, CDCl₃) δ1.41 (9H, s), 3.89 (3H, s), 5.54 (2H, s),7.23-7.26 (2H, m), 7.64 (1H, m), 7.91 (1H, s), 8.00 (1H, s); MS (ES⁺)m/e 400 [MH]⁺. Anal. Found C, 57.36; H, 4.61; N, 24.60%. C₁₉H₁₉F₂N₇Orequires C, 57.14; H, 4.79; N, 24.55%.

EXAMPLE 23-(2,5-Difluorophenyl)-7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine

a) (2-Ethyl-2H-1,2,4-triazol-3-yl)methanol

To a solution of 1,2,4-triazole (10 g, 0.145 mol) in DMF (150 ml) atroom temperature was added sodium hydride (6.4 g of a 60% disp. in oil,0.16 mol) in portions over 15 min. When the addition was complete, thereaction mixture was allowed to cool to room temperature, then cooled inan ice-bath and iodoethane (14 ml, 0.174 mol) was added dropwise over 10mins. The reaction mixture was allowed to warm to room temperature andafter stirring for 3 h the solvents were removed under high vacuum toleave a residue which was partitioned between water (300 ml) and ethylacetate (3×300 ml). The combined organic layers were washed withsaturated brine and dried (MgSO₄), filtered and concentrated undervacuum to leave an oily residue which was purified by distillation (120°C. @˜20 mmHg) to give 1-ethyltriazole contaminated with ˜15% DMF (2.4g). The crude product (2.4 g, 0.025 mol) was dissolved in dry THF (35ml), cooled to −40° C. and n-butyllithium (16.2 ml of a 1.6 molarsolution in hexane, 0.026 mol) was added slowly over 20 mins keeping thetemperature constant. DMF (2.03 ml, 0.026 mol) was then added and after15 mins the reaction mixture was allowed to warm slowly to roomtemperature over 2 h. To the reaction mixture was added methanol (20 ml)followed by sodium borohydride (1 g, 0.026 mol) and the solution wasallowed to stir for 14 h. The solvents were removed under vacuum and theresidue was partitioned between brine (50 ml) and dichloromethane (6×50ml). The combined organic layers were dried (MgSO₄), filtered andconcentrated under vacuum to leave a residue which was purified bysilica gel chromatography using 0-5% methanol in dichloromethane aseluent to give the title compound as an off-white solid (0.5 g, 3%).Data for the title compound: ¹H NMR (250 MHz, CDCl₃) δ1.48 (3H, t, J=7.3Hz), 4.25 (2H, q, J=7.3 Hz), 4.75 (2H, s), 5.14 (1H, br s), 7.78 (1H,s).

b)3-(2,5-Difluorophenyl)-7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine

This compound was prepared using the procedures described in Example 1Steps a), b), c) and d) with (2-ethyl-2H-1,2,4-triazol-3-yl)methanolused instead of (2-methyl-2H-1,2,4-triazol-3-yl)methanol in Step c).Data for the title compound: ¹H NMR (360 MHz, CDCl₃) δ1.40 (9H, s), 1.47(3H, t, J=7.3 Hz), 4.20 (2H, q, J=14.6 & 7.3 Hz), 5.54 (2H, s),7.23-7.27 (2H, m), 7.65 (1H, m), 7.94 (1H, s), 8.00 (1H, s); MS (ES⁺)m/e 414 [MH]⁺. Anal. Found C, 58.17; H, 5.01; N, 23.79%. C₂₀H₂₁F₂N₇Orequires C, 58.10; H, 5.12; N, 23.72%.

EXAMPLE 33-(2,6-Difluorophenyl)-7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine

a)6-Chloro-3-(2,6-difluorophenyl)-7-(1,1-dimethylethyl)-1,2,4-triazolo[4.3-b]pyridazine

A mixture of 3,6-dichloro-4-(1,1-dimethylethyl)pyridazine (2 g, 9.75mmol), 2,6-difluorobenzoic acid hydrazide (2.52 g, 14.6 mmol) (WO95/24403) and triethylamine hydrochloride (2.01 g, 14.6 mmol) in1,4-dioxane (10 ml) was stirred and heated at reflux for 3.5 days. Uponcooling, the volatiles were removed in vacuo and the residue wastriturated with dichloromethane. Any undissolved solid was removed byfiltration. The residue was purified by chromatography on silica geleluting with 0%→25% ethyl acetate in dichloromethane to give therequired product (0.42 g). Data for the title compound: ¹H NMR (250 MHz,CDCl₃) δ1.57 (9H, s), 7.09-7.16 (2H, m), 7.51-7.63 (1H, m), 8.19 (1H,s); MS (ES³⁰) m/e 323 (MH⁺.

b)3-(2,6-Difluorophenyl)-7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-1,2,4-triazolo[4,3-b]pyridazine

This compound was prepared from6-chloro-3-(2,6-difluorophenyl)-7-(1,1-dimethylethyl)-1,2,4-triazolo[4,3-b]pyridazineand (2-ethyl-2H-1,2,4-triazol-3-yl)methanol following the procedure(sodium hydride, DMF) described in WO 98/04559. Data for the titlecompound: m.p. 182° C.; ¹H NMR (400 MHz, CDCl₃) δ1.39-1.45 (12H, m),4.10-4.16 (2H, m), 5.46 (2H, s), 7.09-7.15 (2H, m), 7.52-7.59 (1H, m),7.92 (1H, s), 8.00 (1H, s); MS (ES³⁰) m/e 414 [MH]⁺; Anal. Found: C,58.19; H, 5.05; N, 23.80%. C₂₀H₂₁F₂N₇O requires: C, 58.10; H, 5.12; N,23.72%.

EXAMPLE 47-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2,3,6-trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine

a)6-Chloro-7-(1,1-dimethylethyl)-3-(2,3,6-trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine

2,3,6-Trifluorobenzoyl chloride (4.0 g) was added dropwise to a cooled(15° C.) solution of3-chloro-4-(1,1-dimethylethyl)-6-hydrazinylpyridazine (4 g) in dry1-methyl-2-pyrrolidinone (50 ml). After the addition the reactionmixture was heated at 160° C. for 24 h. The reaction mixture was cooledto room temperature, diluted with ethyl acetate (200 ml), and washedtwice with water (200 ml). The organic phase was separated, dried(sodium sulfate), and evaporated at reduced pressure. The residue wascrystallised from dichloromethane on dilution with diethyl ether toafford the title compound (5.3 g) as a colourless solid: ¹H NMR (400MHz, DMSO-d₆) δ1.52 (9H, s), 7.50 (1H, m), 7.92 (1H, m), 8.45 (1H, s);MS (ES³⁰) m/z 341/343 [MH]⁺.

b) (2-Ethyl-2H-1,2,4-triazol-3-yl)methanol: alternative procedure

1,2,4-Triazole (100.0 g, 1.45 mol) in anhydrous THF (950 ml) was cooledto 0° C. and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (220 g, 1.45 mol)was added in one portion. The reaction mixture was stirred for 30 minuntil complete dissolution was observed. Whilst maintaining theice/water cooling bath, iodoethane (317 g, 2.03 mol) was added dropwiseover a 15 min period resulting in an internal temperature rise to 30° C.The reaction was stirred at room temperature for 16 h, after which theDBU hydroiodide was removed by filtration. The filtrate was cooled to−75° C. under an atmosphere of dry nitrogen. Hexyllithium (458 ml of 33%solution in hexanes) was added dropwise over 25 min keeping the internaltemperature below −55° C. The reaction mixture was aged for 30 min (backto −75° C.) and then dry N,N-dimethylformamide (108 ml, 1.39 mol) wasadded dropwise over 10 min maintaining internal temperature below −60°C. The reaction mixture was aged at −70° C. for 90 min, then allowed towarm to 0° C. over 30 min. Ethanol (340 ml) was added over 10 min.Sodium borohydride (26.3 g, 0.695 mol) was then added portionwisemaintaining the internal temperature below 6° C. After the addition thereaction mixture was allowed to warm to room temperature and stirred for1 h. 2M H₂SO₄ (200 ml) was then added slowly with caution and themixture stirred at room temperature for 20 h. The reaction mixture wasconcentrated to 675 ml and sodium sulfate (135 g) was added in oneportion. The reaction mixture was warmed to 35° C. and stirred for 15min. The solution was extracted with warm (45° C.) isobutyl alcohol(2×675 ml). The combined organic fractions were concentrated underreduced pressure to a volume of approximately 450 ml at which point theproduct crystallised. Heptane (1,125 l) was added and the slurryconcentrated under reduced pressure to remove most of the isobutylalcohol. Heptane was added to give a final slurry volume of 680 ml.After cooling to 0° C., filtration gave the title compound (137 g, 74%from 1,2,4-triazole). ¹H NMR as for Example 2 Step a).

c)7-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2,3,6-trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine

To the product of Step a) (2.60 g) and the foregoing product (1.0 g) indry dimethylsulfoxide (10 ml) was added caesium carbonate (3.05 g), andthe mixture stirred at 50° C. under an atmosphere of dry nitrogen for 24hours. On cooling to room temperature, the mixture was partitionedbetween ethyl acetate and water. The organic phase was separated, washedwith water, evaporated at reduced pressure, and the residuechromatographed on silica gel (eluent 2.5% methanol-dichloromethane).The product was crystallised from ethyl acetate/diethyl ether/isohexane,to afford the title compound as a colourless solid. ¹H NMR (400 MHz,DMSO-d₆) δ1.26 (3H, t, J=7.1 Hz), 1.38 (9H, s), 4.10 (2H, q, J=7.1 Hz),5.53 (2H, s), 7.46 (1H, m), 7.88 (1H, m), 7.94 (1H, s), 8.18 (1H, s); MS(ES⁺) m/z 432 [MH]⁺.

EXAMPLE 57-(1,1-Dimethylethyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2,3,6-trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine

To the product of Example 4 Step a) (2.67 g) and(2-methyl-2H-1,2,4-triazol-3-yl)methanol (prepared as described inEP-A-421210) (0.93 g) in dry dimethylsulfoxide (10 ml) was added caesiumcarbonate (3.14 g), and the mixture stirred at 50° C. under anatmosphere of dry nitrogen for 24 hours. On cooling to room temperature,the mixture was partitioned between ethyl acetate and water. The organicphase was separated, washed with water, evaporated at reduced pressure,and the residue chromatographed on silica gel (eluent 2.5%methanol-dichloromethane). The product was crystallised from ethylacetate/diethyl ether/isohexane, to afford the title compound as acolourless solid. ¹H NMR (500 MHz, DMSO-₆) δ1.39 (9H, s), 3.74 (3H, s),5.50 (2H, s), 7.46 (1H, m), 7.88 (1H, m), 7.90 (1H, s), 8.17 (1H, s); MS(ES⁺) m/z 418 [MH]⁺.

EXAMPLE 67-(1,1-Dimethylethyl)-6-(1-methyl-1H-1,2,4-triazol-3-ylmethoxy)-3-(2,3,6-trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine

To the product of Example 4 Step a) (0.559 g) and(1-methyl-1H-1,2,4-triazol-3-yl)methanol (prepared as described inEP-A-421210) (0.20 g) in dry dimethylsulfoxide (2 ml) was added caesiumcarbonate (0.67 g), and the mixture stirred at 60° C. under anatmosphere of dry nitrogen for 48 hours. On cooling to room temperature,the mixture was partitioned between ethyl acetate and water. The organicphase was separated, washed with water, evaporated at reduced pressure,and the residue chromatographed on silica gel (eluent 2%methanol-dichloromethane). The product was crystallised from ethylacetate/diethyl ether, to afford the title compound as a colourlesssolid. ¹H NMR (400 MHz, DMSO-d₆) δ1.39 (9H, s), 3.85 (3H, s), 5.30 (2H,s), 7.46 (1H, m), 7.86 (1H, m), 8.14 (1H, s) 8.46 (1H, s); MS (ES⁺) m/z418 [MH]⁺.

What is claimed is:
 1. A compound of formula I, or a pharmaceuticallyacceptable salt thereof:

wherein Y represents fluoro and Z represents fluoro; and R¹ representsmethyl or ethyl.
 2. A compound as claimed in claim 1 represented byformula IA, and pharmaceutically acceptable salts thereof:

wherein Y, Z and R¹ are as defined in claim
 1. 3. A compound as claimedin claim 1 represented by formula IIC, and pharmaceutically acceptablesalts thereof:

wherein R¹ is as defined in claim
 1. 4. A compound as claimed in claim 1wherein R¹ represents methyl.
 5. A compound as claimed in claim 1wherein R¹ represents ethyl.
 6. A compound selected from:7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2,3,6-trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine;7-(1,1-dimethylethyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2,3,6-trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine;7-(1,1-dimethylethyl)-6-(1-methyl-1H-1,2,4-triazol-3-ylmethoxy)-3-(2,3,6-trifluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine;and pharmaceutically acceptable salts thereof.
 7. A pharmaceuticalcomposition comprising a compound of formula I as defined in claim 1 ora pharmaceutically acceptable salt thereof in association with apharmaceutically acceptable carrier.
 8. A process for the preparation ofa compound as claimed in claim 1, which comprises: (A) reacting acompound of formula III with a compound of formula IV:

 wherein Y, Z and R¹ are as defined in claim 1, and L¹ represents asuitable leaving group; or (B) reacting a compound of formula XI (or its1,2,4-triazolo[4,3-b]pyridazin-6-one tautomer) with a compound offormula XII:

 wherein Y, Z and R¹ are as defined in claim 1, and L³ represents asuitable leaving group; or (C) reacting trimethylacetic acid with acompound of formula XIII:

 wherein Y, Z and R¹ are as defined in claim 1; in the presence ofsilver nitrate and ammonium persulphate; or (D) reacting a compound offormula XIV with a compound of formula XV:

 wherein Y, Z and R¹ are as defined in claim 1, M represents —B(OH)₂ or—Sn(Alk)₃ in which Alk represents a C₁₋₆ alkyl group, and L⁴ representsa suitable leaving group; in the presence of a transition metalcatalyst.
 9. A method for the treatment and/or prevention of anxietywhich comprises administering to a patient in need of such treatment aneffective amount of a compound of formula I as defined in claim 1 or apharmaceutically acceptable salt thereof.